DESCRIPTION (From the applicant's abstract): Gamma-secretase catalyzed cleavages are of interest for a number of reasons. First, gamma-secretase is an unusual proteolytic activity with the cleavage site of the substrate predicted to lie within the transmembrane domain. Moreover, position of the gamma-cleavage site with respect to the membrane, not the primary amino acid sequence, appears to be the major determinant of cleavage, with the length of the lumenal transmembrane domain (TMD) determining that position. Whether gamma-secretase actually cleaves residues within the membrane is a subject of much controversy. To date, there is no evidence showing that a protease can cleave bonds buried within a membrane. Second, altered gamma-secretase cleavage is implicated in the development of AD. With the exception of the APPK670N,M67IL mutation, all FAD-linked mutations alter gamma-secretase activity by increasing the amount of the more amyloidogenic Abeta species, Abeta 42. Third, APP carboxyl terminal fragments (CTF) do not appear to be the only substrate for gamma-secretase activity. The cell surface receptor Notch undergoes processing analogous to APP. Several sequential cleavages in the lumenal/extracellular domain are followed by a final cleavage of residues near the cytoplasm/membrane junction mediated by a gamma-secretase like cleavage. Finally, because gamma-secretase cleavage is the final step in the generation of Abeta, it remains a major therapeutic target for strategies designed to lower Abeta production. Thus, gamma-secretase is not only an unusual proteolytic event, but its activity has important biological consequences both in the normal state and with respect to pathogenesis and treatment of AD. To further characterize and ultimately identify the proteases responsible for the gamma-secretase activity we propose to: 1. Map the position of the gamma-secretase site with respect to the membrane in full length APP versus an APP CTF using biochemical methods and then determine whether mutations that alter gamma-secretase cleavage alter membrane positioning of APP CTF. 2. Determine the relative potency of gamma-secretase inhibitors using an in vitro gamma-secretase activity assay. 3. Purify gamma-secretase using affinity based approaches to identify candidate candidate gamma-secretases that will then be screened for gamma-activity by sense and antisense expression studies.